1. Field of the Invention
This invention relates to a method for labeling DNA and RNA, and more specifically, this invention relates to a method for labeling DNA and RNA using radical-producing chemical agents.
2. Background of the Invention
DNA microchip technology is a rapid, high throughput platform for nucleic acid hybridization reactions. However, nucleic acid fragmentation and labeling are two of the limiting steps in the development of rapid protocols for DNA microchip technology.
PCR and other amplification techniques are utilized for bacteria identification. Immunological methods and mass-spectrometry also have been adapted for this purpose, but are expensive and cumbersome.
Several enzymatic and chemical protocols are available for fluorescent labeling of nucleic acids. All of these methods are expensive and time consuming. Most of these protocols demand careful prerequisite nucleic acid isolation, fractionation (generally requiring one or more hours), labeling, separate sample fragmentation procedures and a final purification step.
Typical nucleic acid labeling methods adopt a myriad of approaches. For example, M. D. Schena et al., Science 270, 467–470 (1995); J. L. DeRisi et al., Science 278, 680–686 (1997); G. P. Yang et al., Nucl. Acid Res. 27, 1517–1523 (1999); K. Wang et al., Gene 229, 101–108 (1999), and M. Wilson et al. Proc. Natl. Acad. Sci USA 96, 12833–12838 all rely on effecting labeling using reverse transcriptase. Typically, this process requires from one to two hours to complete.
D. Guiliano et al. BioTechniques 27 146–152 (1999) and G. T. Hermanson, Bioconjugate Techniques (Academic Press, Inc. San Diego, Calif., 1996) utilize random priming. However, these protocols require from 3 to 10 hours to complete.
Terminal transferase protocols are featured in K. L. Gunderson et al. Genome Res. 8, 1142–1153 (1998) and L. Wodicka et al. Nat. Biotechnol. 15, 1359–1367. However, these processes also require between 1 and 2 hours to run.
Polymerase Chain Reaction (PCR) protocols for labeling are widespread. Typical references for PCR processes include R. J. Sapolsky et al. Genomics 33, 445 –456 (1996); M. T. Cronin et al. Hum. Mutat. 7, 244–255 (1996); S. Tyagi et al. Nat. Biotechnol 16, 49–53 (1998); and P. N. Gilles et al. Nat. Biotechnol 17, 365–370 (1999). However, PCR protocols require between 1 and 2 hours to complete.
A need exists in the art for a simple protocol for labeling nucleic acids found either in DNA or RNA. The protocol should require mild conditions of reaction and should yield high amounts of cross-linked complexes in short incubation times. The method should facilitate both the labeling and fragmentation at random sites of nucleic acids, therefore being independent of sequence or two dimensional structures. The method should facilitate the end-labeling of nucleic acids and further accommodate a broad number of label derivatives, the later to be attached to nucleic acids. Lastly, the method should accommodate automated processes.